ABSTRACT
BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.
Subject(s)
Autocrine Communication , Immunoglobulin E/immunology , Inflammation/immunology , Inflammation/metabolism , Mast Cells/immunology , Mast Cells/metabolism , Tachykinins/metabolism , Anaphylaxis/immunology , Anaphylaxis/metabolism , Animals , Disease Models, Animal , Female , Interleukin-6/biosynthesis , Lung/immunology , Lung/metabolism , Lung/pathology , Mice , Mice, Knockout , Receptors, IgE/metabolism , Receptors, Neurokinin-1/metabolism , Signal Transduction , Tumor Necrosis Factors/biosynthesisABSTRACT
Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.
Subject(s)
Dendritic Cells/immunology , Immunity, Cellular/immunology , Interleukin-12/immunology , Receptors, Neurokinin-1/immunology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/immunology , Cyclic AMP Response Element-Binding Protein/metabolism , Dendritic Cells/metabolism , Dendritic Cells/transplantation , Flow Cytometry , Immunization/methods , Immunophenotyping , Interleukin-10/immunology , Interleukin-10/metabolism , Interleukin-12/genetics , Interleukin-12/metabolism , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Multiprotein Complexes/immunology , Multiprotein Complexes/metabolism , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/metabolism , Signal Transduction/immunology , TOR Serine-Threonine Kinases/immunology , TOR Serine-Threonine Kinases/metabolismABSTRACT
Aberrant activation of macrophages in arterial walls by oxidized lipoproteins can lead to atherosclerosis. Oxidized lipoproteins convert macrophages to foam cells through lipid uptake and TLR signaling. To investigate the relative contributions of lipid uptake and TLR signaling in foam cell formation, we established an in vitro assay using liposomes of defined lipid compositions. We found that TLRs signaling through Toll/IL-1R domain-containing adapter inducing IFN-ß promoted foam cell formation by inducing both NF-κB signaling and type I IFN production, whereas TLRs that do not induce IFN, like TLR2, did not enhance foam cell formation. Addition of IFN-α to TLR2 activator promoted robust foam cell formation. TLR signaling further required peroxisome proliferator-activated receptor α, as inhibition of peroxisome proliferator-activated receptor α blocked foam cell formation. We then investigated the ability of endogenous microparticles (MP) to contribute to foam cell formation. We found that lipid-containing MP promoted foam cell formation, which was enhanced by TLR stimulation or IFN-α. These MP also stimulated foam cell formation in a human skin model. However, these MP suppressed TNF-α production and T cell activation, showing that foam cell formation can occur by immunosuppressive MP. Taken together, the data reveal novel signaling requirements for foam cell formation and suggest that uptake of distinct types of MP in the context of activation of multiple distinct TLR can induce foam cell formation.
Subject(s)
Cell Differentiation/immunology , Cell-Derived Microparticles/immunology , Foam Cells/immunology , Foam Cells/metabolism , Macrophages/immunology , Toll-Like Receptors/metabolism , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Line, Tumor , Cell-Derived Microparticles/metabolism , Cell-Derived Microparticles/pathology , Cells, Cultured , Foam Cells/pathology , HeLa Cells , Humans , Ligands , Lipid Metabolism/immunology , Macrophages/metabolism , Macrophages/pathology , Melanoma, Experimental/immunology , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/immunology , Toll-Like Receptors/physiologyABSTRACT
Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.
Subject(s)
Cell Communication , Dendritic Cells/metabolism , Endosomes/metabolism , Exosomes/genetics , MicroRNAs/physiology , Animals , Antigen Presentation , Biomarkers/metabolism , Cytosol/metabolism , Dendritic Cells/cytology , Exosomes/metabolism , Gene Expression Profiling , Membrane Fusion , Mice , Oligonucleotide Array Sequence AnalysisABSTRACT
The prevailing idea regarding the mechanism(s) by which therapeutic immunosuppressive dendritic cells (DCs) restrain alloimmunity is based on the concept that they interact directly with antidonor T cells, inducing anergy, deletion, and/or regulation. However, this idea has not been tested in vivo. Using prototypic in vitro-generated maturation-resistant (MR) DCs, we demonstrate that once MR-DCs carrying donor antigen (Ag) are administered intravenously, they decrease the direct and indirect pathway T-cell responses and prolong heart allograft survival but fail to directly regulate T cells in vivo. Rather, injected MR-DCs are short-lived and reprocessed by recipient DCs for presentation to indirect pathway CD4(+) T cells, resulting in abortive activation and deletion without detrimental effect on the number of indirect CD4(+) FoxP3(+) T cells, thus increasing the regulatory to effector T cell relative percentage. The effect on the antidonor response was independent of the method used to generate therapeutic DCs or their viability; and in accordance with the idea that recipient Ag-presenting cells mediate the effects of therapeutic DCs in transplantation, prolongation of allograft survival was achieved using donor apoptotic MR-DCs or those lacking surface major histocompatibility complex molecules. We therefore conclude that therapeutic DCs function as Ag-transporting cells rather than Ag-presenting cells to prolong allograft survival.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/transplantation , Animals , Antigen Presentation , Base Sequence , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation , DNA Primers/genetics , Dendritic Cells/cytology , Immunosuppression Therapy , Injections, Intravenous , Isoantigens , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Tissue Donors , Transplantation, HomologousABSTRACT
Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However, whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin, where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6, which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs, purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless, these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Inflammation Mediators/physiology , Interleukin-17/physiology , Skin/immunology , Skin/metabolism , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Cell Culture Techniques , Cell Differentiation/immunology , Cell Movement/immunology , Coculture Techniques , Dendritic Cells/pathology , Humans , Interleukin-15/biosynthesis , Interleukin-15/physiology , Interleukin-6/metabolism , Interleukin-6/physiology , Langerhans Cells/immunology , Langerhans Cells/metabolism , Lymphocyte Culture Test, Mixed , Organ Culture Techniques , Signal Transduction/immunology , Skin/pathology , T-Lymphocytes, Helper-Inducer/pathology , Th1 Cells/immunology , Th1 Cells/metabolism , Th1 Cells/pathologyABSTRACT
Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.
Subject(s)
Dendritic Cells/drug effects , Immunity, Cellular/drug effects , Inflammation Mediators/pharmacology , Receptors, Neurokinin-1/physiology , Tachykinins/pharmacology , Adoptive Transfer , Animals , Apoptosis/drug effects , Apoptosis/immunology , Bone Marrow Cells/metabolism , CD40 Antigens/metabolism , CD40 Antigens/physiology , Cell Survival/drug effects , Cell Survival/genetics , Dendritic Cells/metabolism , Dendritic Cells/physiology , Dendritic Cells/transplantation , Enzyme Activation/drug effects , Immunity, Cellular/genetics , Immunity, Cellular/physiology , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/genetics , Receptors, Neurokinin-1/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Tachykinins/metabolismABSTRACT
Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.